Creation of a Biological Atrioventricular Bypass to Compensate for Atrioventricular Block

ABSTRACT

A method of creating an atrioventricular bypass tract for a heart comprises growing mesenchymal stem cells into a strip with two ends, attaching one end of the strip onto the atrium of the heart, and attaching the other end of the strip to the ventricle of the heart, to create a tract connecting the atrium to the ventricle to provide a path for electrical signals generated by the sinus node to propagate across the tract and excite the ventricle.

This application claims the benefit of, and priority from, U.S.Provisional Application No. 60/532,363, filed Dec. 24, 2003.

The invention disclosed herein was made at least in part with funding bythe U.S. Government, specifically the USPHS, and NHLBI under grantnumber HL-28958. Therefore, the U.S. Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced to asfootnotes or within parentheses. Disclosures of these publications intheir entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thisinvention pertains. Full bibliographic citations for these referencesmay be found at the end of this application, preceding the claims.

One of the major indications for electronic pacemaker therapy is highdegree heart block, such that a normally functioning sinus node impulsecannot propagate to the ventricle. The result is ventricular arrestand/or fibrillation, and death.

Acute myocardial infarction (MI) afflicts millions of people each yearinducing significant mortality and, in a large number of survivors,marked reductions in myocyte number and in cardiac pump function. Adultcardiac myocytes divide only rarely, and the usual response to myocytecell loss is hypertrophy that often progresses to congestive heartfailure, a disease with a significant annual mortality. There have beenrecent reports of the delivery of mesenchymal stem cells (MSCs amultipotent cell population of blood lineage) to the hearts of post-Mipatients resulting in improved mechanical performance^(1,2). Thepresumption in these and other animal studies³, is that the MSCsintegrate into the cardiac syncytium and then differentiate into newheart cells restoring mechanical function.

SUMMARY OF THE INVENTION

The present invention uses biological means for cell therapy to build abypass tract in the heart that will take over the function of a diseasedatrioventricular node. Adult human mesenchymal stem cells (hMSCs) may beprepared in one of four ways (see below) and grown in culture on anon-bioreactive material. Once growth is complete the material has oneend sutured to the atrium, and the other to the ventricle. Electricalsignals generated by the sinus node to activate the atria will propagateacross the artificially constructed tract to excite the ventricle aswell. In this way the normal sequence of atrioventricular activationwill be maintained.

Four methods that may be used for preparing the hMSCs are:

1: In culture without incorporation of additional molecular determinantsof conduction. Here the cells' own characteristic to generate gapjunctions that communicate electrical signals are used as a means topropagate an electronic wave from atrium to ventricle.

2: In culture following electroporation to add the gene for connexins43, 40 and/or 45, the culture's electrotonic propagation of atrialsignals to the ventricle.

3: In culture following electroporation to add the alpha and theaccessory subunits of the L-type calcium channel, thereby increasing thelikelihood of not just electrotonic propagation of a wavefront, but itsactive propagation by an action potential.

4: A combination of 2 and 3.

The preparation of a bypass in this fashion not only will facilitatepropagation from atrium to ventricle, but will provide sufficient delayfrom atrial to ventricular contraction to maximize ventricular fillingand emptying. The goal is to mimic the normal activation and contractilesequence of the heart. Moreover, this approach, when used with genetherapy and stem cell technology Lo improve atrial impulse initiation inthe setting of sinus node disease offers a completely physiologic systemrather than its electronic replacement.

According to the invention, a method of creating an atrioventricularbypass tract for a heart is provided, comprising growing mesenchymalstem cells into a strip with two ends, attaching one end of the striponto the atrium of the heart, and attaching the other end of the stripto the ventricle of the heart, to create a tract connecting the atriumto the ventricle to provide a path for electrical signals generated bythe sinus node to propagate across the tract and excite the ventricle.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Identification of connexins in gap junctions of hMSCs.Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, Immunoblotanalysis of Cx43 in canine ventricle myocytes and hMSCs. Whole celllysates (120 jig) from ventricle cells or hMSCs were resolved by SDS,transferred to membranes, and blotted with Cx43 antibodies. Migration ofmolecular weight markers is indicated to the right to the blot.

FIG. 2. Macroscopic and single channel properties of gap junctionsbetween hMSC pairs. Gap junction currents (Ij) elicited from hMSCs usingsymmetrical bipolar pulse protocol showed two types of voltage dependentcurrent deactivation: (A)-symmetrical, (B): asymmetrical.

C,D Single channel recordings from pairs of hMSCs. Pulse protocol (V₁and V₂) and associated multichannel currents (Iz) recorded from a cellpair during maintained V_(j) of ±80 mV. The discrete current stepsindicate the opening and closing of single channels. Dashed line: zerocurrent level. The all points current histograms on the right-hand siderevealed a conductance of ˜50 pS. Glass coverslips with adherent cellswere transferred to an experimental chamber perfused at room temperature(˜22° C.) with bath solution containing (mM): NaCI, 150; KCl, 10; CaCl₂,2; HEPES, 5 (pH 7.4); glucose, 5. The patch pipettes were filled withsolution containing (mM): K⁺ aspartate⁻, 120; NaCI, 10; MgATP, 3; HEPES,5 (pH 7.2); EGTA, 10 (pCa˜8); filtered through 0.22 μm pores. Whenfilled, the resistance of the pipettes measured 1-2 MΩ. Experiments werecarried out on cell pairs using a double voltage-clamp. This methodpermitted to control the membrane potential (V_(m)) and measure theassociated junctional currents (Ij).

FIG. 3. Macroscopic properties of junctions in cell pairs between a hMSCand HeLa cell expressing only Cx40, Cx43 or Cx45. In all cases hMSC toHela cell coupling was tested 6 to 12 after hours intiating co-culture.

A, Ij elicited in response to a series of voltage steps (V_(j)) inhMSC-HeLaCx43 pairs.

Top: symmetrical current deactivation; bottom: asymmetrical currentvoltage dependence.

B, Macroscopic Ij recordings from hMSC-HelaCx40 pairs exhibitsymmetrical (top panel) and asymmetrical (bottom panel) voltagedependent deactivation.

C, Asymmetric Ij from hMSC-HeLaCx43 pair exhibits voltage dependentgating when Cx45 side is relative negative. Ij recorded from hMSC.

D, Cell-to-cell. LY spread in cell pairs: from a HeLa Cx43 to an hMSC(top panel) and from an hMSC to a HeLa Cx43 to (bottom panel). In bothcases a pipette containing 2 mM LY was attached to the left-handed cellin the whole-cell configuration.

Epifluorescent micrographs taken at 12 min after dye injection show LYspread to the adjacent (right-handed) cell. The simultaneously measuredjunctional conductance⁶ revealed g_(j) of ˜16 nS and ˜18 nS of thepairs, respectively. Cell Tracker green was used to distinguish hMSCsfrom HeLa cells or vice versa in all experiments⁸.

FIG. 4. Macroscopic and single channel properties of gap junctionsbetween hMSC-canine ventricle cell pairs. Myocytes were plated between12 and 72 hours and co-cultured with hMSCs for 6 to 12 hours beforemeasuring coupling. A, Top panel: Phase-contrast micrograph of ahMSC-canine ventricle pair. Bottom pane: Monopolar pulse protocol (V₁and V₂) and associated macroscopic junctional currents (Iz) exhibitingasymmetrical voltage dependence. B, Top panel: Multichannel currentelicited by symmetrical biphasic 60 mV pulse. Dashed line, zero currentlevel; dotted lines, represent discrete current steps indicative ofopening and closing of channels. The current histograms yielded aconductance of ˜40-50 pS. Bottom panel: Multichannel recording duringmaintained V_(j) of 60 mV. The current histograms revealed severalconductances of 48 to 64 pS with several events with conductance of 84pS to with 99 pS (arrows) which resemble operation of Cx43, heterotypicCx40-Cx43 and/ or homotypic Cx40 channels.

DESCRIPTION OF THE INVENTION

According to the invention, a method of creating an atrioventricularbypass tract for a heart is provided, comprising growing mesenchymalstem cells into a strip with two ends, attaching one end of the striponto the atrium of the heart, and attaching the other end of the stripto the ventricle of the heart, to create a tract connecting the atriumto the ventricle to provide a path for electrical signals generated bythe sinus node to propagate across the tract and excite the ventricle.

The steps of attaching may be performed by suturing. The stem cells maybe adult human mesenchymal stem cells. The step of growing may comprisegrowing the stem cells in culture on a non-bioreactive material. Thestep of growing may be performed in an environment substantially free ofany additional molecular determinants of conduction.

The method may further comprise a step of adding a gene to themesenchymal stem cells by electroporation. The gene may encode for aconnexin, such as connexin 40, connexin 43, and/or connexin 45. The stepof adding a gene by electroporation may include adding alpha andaccessory subunits of L-type calcium. The step of adding a gene byelectroporation may include adding the gene for connexions and addingalpha and accessory subunits of L-type calcium channel.

MSCs express connexins that are the building block proteins of gapjunctions and can form functional gap junctions with one another, withcell lines expressing cardiac connexins, and with adult cardiacmyocytes. Further, the connexins expressed suggest that hMSCs shouldreadily integrate into electrical syncytia of many tissues promotingrepair or serving as the substrate for a therapeutic delivery system.

Human mesenchymal stem cells (Poietics™ hMSCs—Mesenchymal stem cells,Human Bone Marrow) were purchased from Clonetics/BioWhittaker(Walkersville, Md.) and cultured in MCS growing media and used frompassages 2-4. Typical punctate staining for Cx43 and Cx40 was seen alongregions of intimate cell to cell contact of the MSCs grown in culture asmonolayers (FIG. 1 A,B). Cx45 staining was also detected but unlike thatof Cx43 or Cx40 was not typical of connexin distribution in cells.Rather it was characterized by fine granular cytoplasmic andreticular-like staining with no readily observed membrane associatedplaques (FIG. 1C). This does not exclude the possibility that Cx45channels exist but does imply that their number relative to Cx43 andCx40 homotypic, heterotypic and heteromeric channels is low. FIG. 1Dillustrates Western blot analysis⁴ for canine ventricle myocytes andhMSCs with a Cx43 polyclonal antibody which adds further proof of Cx43presence in hMSCs.

Gap junctional coupling among hMSCs is demonstrated in FIG. 2.Junctional currents recorded between hMSC pairs show quasi-symmetrical(FIG. 2A) and asymmetrical (FIG. 2B) voltage dependency arising inresponse to symmetric transjunctional voltage steps of equal amplitudebut opposite sign. These behaviors are typically observed in cells whichco-express Cx43 and Cx40⁴.

FIGS. 2C and 2D illustrate typical multichannel recordings from a hMSCpair. Using 120 mM K aspartate as a pipette solution channels wereobserved with unitary conductances of 28-80 pS range. Operation ofchannels with ˜50 pS conductance (see FIG. 2 C) is consistent withpreviously published values^(5,6) for Cx43 homotypic channels. This doesnot preclude the presence of other channel types, it merely suggeststhat Cx43 forms functional channels in hMSCs.

To further define the nature of the coupling hMSCs were co-cultured withhuman HeLa cells stably transfected with Cx43, Cx40, and Cx45⁷ and itwas found that hMSCs were able to couple to all these transfectants.FIG. 3A shows an example of junctional currents recorded between an hMSCand HeLaCx43 cell pairs that manifested symmetrically and asymmetricallyvoltage dependent currents. The quasi-symmetric record suggests that thedominant functional channel is homotypic Cx43 while the asymmetricrecord suggests the activity of another connexin in the hMSC (presumablyCx40 as shown by immunohistochemistry, see FIG. 1) that could be eithera heterotypic or heteromeric form or both. These records are similar tothose published for transfected cells: heterotypic and mixed(heteromeric) forms of Cx40 and Cx43^(4,8). Co-culture of hMSCs withHeLa cells transfected with Cx40 (FIG. 3B) also revealed symmetric andasymmetric voltage dependent junctional currents consistent with theco-expression of Cx43 and Cx40 in the hMSCs similar to the data for Cx43HeLa-hMSC pairs. HeLa cells transfected with Cx45 coupled to hMSCsalways produced asymmetric junctional currents with pronounced voltagegating when Cx45 (HeLa) side was negative (FIG. 3C). This is consistentwith the dominant channel forms in the hMSC being Cx43 and Cx40 as bothproduce asymmetric currents when they form heterotypic channels withCx45^(4,8). This does not exclude Cx45 as a functioning channel in hMSCsbut it does indicate that Cx45 is a minor contributor to cell to cellcoupling in hMSCs.

The lack of visualized plaques in the immunostaining for Cx45 (FIG. 1)further supports this interpretation.

FIG. 3D shows Lucifer Yellow transfer from HeLaCx43 cell to an hMSC cell(top panel) and transfer from an hMSC to a HeLaCx43 (bottom panel). Thejunctional conductance of the cell pairs was simultaneously measured bymethods described earlier⁶ and revealed conductances of ˜16 nS and ˜18nS, respectively. The transfer of Lucifer Yellow was similar to thatpreviously reported for homotypic Cx43 or co-expressed Cx43 and Cx40 inHeLa cells⁶. Cell Tracker green (Molecular Probes) was always used inone of the two populations of cells to allow heterologous pairs to beidentified⁸.

hMSCs were also co-cultured with adult canine ventricular myocytes. Asshown in FIG. 4 the hMSCs couple electrically with cardiac myocytes.Both macroscopic (FIG. 4A) and multichannel (FIG. 4B) records wereobtained. Junctional currents in FIG. 4A are asymmetric while those inFIG. 4B show unitary events of the size range typically resulting fromthe operation of homotypic Cx43 or heterotypic Cx43-Cx40 or homotypicCx40 channels^(4,8). Heteromeric forms are also possible whoseconductances are the same or similar to homotypic or heterotypic forms.

In studies of cell pairs were demonstrated effective coupling of hMSC toother hMSC (13.8±2.4 nS, n=14), to HeLa Cx43 (7.9±2.1 ns, n=7), to HeLaCx40 (4.6±2.6 nS, n=5), to HeLa Cx45 (11±2.6 nS, n=5) and to ventricularmyocytes (1.5±1.3 nS, n=4). Results show that hMSCs couple to oneanother via Cx43 and Cx40. In addition, they form functional gapjunction channels with cells transfected with Cx43, Cx40 or Cx45 as wellas canine ventricular cardiomyocytes. These data support the possibilityof using MSCs as a therapeutic substrate for repair of cardiac tissue.Other syncytia such as vascular smooth muscle or endothelial cellsshould also be able to couple to the hMSCs because of the ubiquity ofCx43 and Cx40^(9,10). Thus they may also be amenable to hMSCs basedtherapeutics, as follows: hMSCs can be transfected to express ionchannels which then can influence the surrounding synctial tissue.

Alternatively, the hMSCs can be transfected to express genes thatproduce small therapeutic molecules capable of permeating gap junctionsand influencing recipient cells. Further, for short term therapy, thesmall molecules can be directly loaded into hMSCs for delivery torecipient cells. The success of such an approach is dependent on gapjunction channels as the final conduit for delivery of the therapeuticagent to the recipient cells. The feasibility of one such approach wasdemonstrated by transfecting hMSCs with mHCN2, a gene encoding thecardiac pacemaker channel, and delivering them to the canine heart wherethey generate a spontaneous rhythm.

REFERENCES

-   1. Strauer, B. E. et al Repair of infarcted myocardium by autologous    intracoronary mononuclear bone marrow cell transplantation in    humans. Circulation 106, 1913-1918(2002).-   2. Perin, E. C., Geng, Y. J. & Willerson, J. T. Adult stem cell    therapy in perspective. Circulation 107, 935-938 (2003).-   3. Orlic, D. et al. Bone marrow cells regenerate infarcted    myocardium. Nature 410, 701-705 (2001).-   4. Valiunas, V., Gemel, J., Brink, P. R. & Beyer, E. C. Gap junction    channels formed by coexpressed connexin40 and connexin43. Am. J.    Physiol. Heart Circ. Physiol. 2001. October; 281. (4.):H1675. -89.    281, H1675-H1689 (2001).-   5. Valiunas, V., Bukauskas, F. F. & Weingart, R. Conductances and    selective permeability of connexin43 gap junction channels examined    in neonatal rat heart cells. Circ. Res. 80, 708-719 (1997).-   6. Valiunas, V., Beyer, E. C. & Brink, P. R. Cardiac gap junction    channels show quantitative differences in selectivity. Circ. Res.    91, 104-111 (2002).-   7. Elfgang,C. et al. Specific permeability and selective formation    of gap junction channels in connexin-transfected HeLa cells. J. Cell    Biol. 129, 805-817 (1995).-   8. Valiunas. V., Weingart. R. & Brink. P. R. Formation of    heterotypic gap junction channels by connexins 40 and 43. Circ.    Res. 2000. February 4;86. (2.) :E42.-9.86,E42-E49 (2000).-   9. Wang. H. Z. et al. Intercellular communication in cultured human    vascular smooth muscle cells. Am. J. Physiol Cell Physiol 281,    C75-C88 (2001).-   10. Beyer. E. C. Gap junctions. Int. Rev. Cytol. 137C, 1-37 1993).

1. A method comprising growing mesenchymal stem cells into a striphaving two ends, whereby one end of the strip may be attached to theventricle of a heart and the other end may be attached to the atrium ofthe heart to create an atrioventricular bypass tract that provides apath for electrical signals generated by the sinus node to propagateacross the tract and excite the ventricle.
 2. The method of claim 1,wherein the stem cells are adult human mesenchymal stem cells.
 3. Themethod of claim 1, wherein the stem cells are grown in culture on anon-bioactive material.
 4. The method of claim 1, wherein the stem cellsare grown in an environment substantially free of additional moleculardeterminants of conduction.
 5. The method of claim 1, wherein the cellsare transfected to express a nucleic acid encoding a protein or peptideor biologically active fragment thereof.
 6. The method of claim 5,wherein the nucleic acid encodes a connexin.
 7. The method of claim 6,wherein the connexin is connexin
 40. 8. The method of claim 6, whereinthe connexin is connexin
 43. 9. The method of claim 6, wherein theconnexin is connexin
 45. 10. The method of claim 5, wherein the nucleicacid encodes an alpha and accessory subunits of the L-type calciumchannel or biologically active fragment thereof.
 11. The method of claim5, further comprising transfecting the cells with a nucleic acidencoding an alpha and accessory subunits of the L-type calcium channelor biologically active fragments thereof.
 12. An atrioventricular bypasstract for a heart that provides a path for electrical signals generatedby the sinus node to propagate across the tract and excite theventricle, comprising mesenchymal stem cells organized into a striphaving two ends, whereby one end of the strip may be attached to theventricle of a heart and the other end may be attached to the atrium ofthe heart thereby creating the atrioventricular bypass tract.
 13. Thebypass tract of claim 12, wherein the stem cells are adult humanmesenchymal stem cells.
 14. The bypass tract of claim 12, wherein thestem cells are grown in culture on a non-bioactive material.
 15. Thebypass tract of claim 12, wherein the stem cells are grown in anenvironment substantially free of additional molecular determinants ofconduction.
 16. The bypass tract of claim 12, wherein the stem cells aretransfected with a nucleic acid encoding a protein or peptide orbiologically active fragment thereof.
 17. The bypass tract of claim 16,wherein the nucleic acid encodes a connexin.
 18. The bypass tract ofclaim 17, wherein the connexin is connexin
 40. 19. The bypass tract ofclaim 17, wherein the connexin is connexin
 43. 20. The bypass tract ofclaim 17, wherein the connexin is connexin
 45. 21. The bypass tract ofclaim 16, wherein the nucleic acid encodes an alpha and accessorysubunits of the L-type calcium channel or biologically active fragmentsthereof.
 22. The bypass tract of claim 16, further comprisingtransfecting the cells with a nucleic acid encoding an alpha andaccessory subunits of the L-type calcium channel or biologically activefragments thereof.
 23. The bypass tract of claim 16, wherein the nucleicacid encodes a hyperpolarization-activated cyclic nucleotide gated (HCN)channel.
 24. The bypass tract of claim 23, wherein the HCN channel isHCN2.
 25. The method of claim 6, wherein the nucleic acid encodes ahyperpolarization-activated cyclic nucleotide gated (HCN) channel. 26.The method of claim 25, wherein HCN channel is HCN2.